Christine Blattner

P300 is an important transcriptional co-factor. By stimulating the transfer of acetyl residues onto histones and several key transcription factors, p300 enhances transcriptional initiation and impacts cellular processes including cell proliferation and cell division. Despite its importance for cellular homeostasis, its regulation is poorly understood. We show that TRIM25, a member of the TRIM protein family, targets p300 for proteasomal degradation. However, despite TRIM25’s RING domain and E3 activity, degradation of p300 by TRIM25 is independent of TRIM25-mediated p300 ubiquitination. Instead, TRIM25 promotes the interaction of p300 with dynein, which ensures a microtubule-dependent transport of p300 to cellular proteasomes. Through mediating p300 degradation, TRIM25 affects p300-dependent gene expression.

X15695 stabilizes p53. Western blot analysis of p53 in extracts of MCF-7 (A) and T47D (B) cells treated with vehicle or the indicated concentrations of X15695 for 48 hours. Anti-β-actin antibody was used for the loading control (same as it appears in Fig. 3A and B). Quantitative RT-PCR of p53 expression in MCF-7 (C) and T47D (D) cells after treatment with vehicle (DMSO) or the indicated concentrations of X15695 for 48 hours. The results are the mean ± SEM (n = 3; ns, nonsignificant). E, Western blot analysis of p53 in extracts of MCF-7 cells treated with 0.5 μmol/L MG132 in the presence and absence of 1 μmol/L X15695 for 48 hours. Anti-β-actin antibody was used for the loading control. F, Quantification of the p53 protein band intensities relative to the intensity of the β-actin signal. The results are the means ± SEM (n = 3; *, P ≤ 0.05; ns, nonsignificant result). Western blot analysis of MDM2 levels in extracts of MCF-7 (G) and T47D (H) cells after treatment with the indicated concentration of X15695 for 48 hours. Anti-β-actin antibody was used for determination of equal protein level. The protein band intensities were quantified and presented below. Measurement of DCF fluorescence in MCF-7 (I) and T47D (J) cells treated with the indicated concentrations of X15695 or DMSO for 48 hours. The signals were normalized to DNA content of the samples measured after Hoechst 33258 staining. The results are the mean ± SEM (n = 3; ***, P ≤ 0.001; ****, P ≤ 0.0001). K, Reduced H2DCF-DA oxidation assay showing the effect of NAC on ROS production in MCF-7 cells treated with X15695 for 48 hours. Results represent the mean H2DCF-DA oxidation normalized to DNA content (n = 1 with 6 technical replicates). L, Western blots of lysates of MCF-7 cells treated with the indicated concentrations of NAC 1 hour prior to treatment with 1 μmol/L X15695 or DMSO for 48 hours. Expression of p53 was monitored using anti-p53 antibody and anti-β-actin antibody was used as a protein loading control. M and N, Immunofluorescence images showing p53 localization in MCF-7 and T47D cells treated with vehicle (DMSO) and 1 μmol/L X15695 for the indicated time periods. For staining, anti-p53 antibody was used followed by goat anti-mouse IgG, Alexa Fluor 488 antibody – green) and DAPI (magenta; M) or Alexa Fluor 488 antibody – red) and DAPI (light blue; N).

X15695 and fulvestrant downregulate ERα level and proliferation of breast cancer cell proliferation A, Western blot analysis of ERα in extracts of MCF-7 cells treated for the indicated timepoints with 1 μmol/L of fulvestrant or X15695 or DMSO (vehicle). β-actin was used for the loading control. The protein signals were quantified and presented in percentages as the signal of ERα relative to the β-actin signal (right). The value at zero time of treatment was set to 100%. The values are the mean ± SEM (n = 3; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). B, Western blot analysis of ERα in extracts of MCF-7 cells treated with the indicated concentration of fulvestrant or X15695 for 8 hours. β-actin was used for the loading control. The protein signals were quantified and presented in percentages relative to the vehicle-treated sample (right). The values are the mean ± SEM (n = 3; ****, P ≤ 0.0001). C, Measurement of cell viability by MTT assay of tamoxifen resistant cells (TRMCF-7) treated with the indicated concentrations of X15695, fulvestrant, and tamoxifen for 48 hours. Five replicates were used for each experiment, and the experiment was repeated three times (**, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001; ns, nonsignificant). D, Representative image of a clonogenic assay showing the effect of the indicated concentrations of X15695, fulvestrant, and tamoxifen on clonal expansion of tamoxifen-resistant MCF-7 cells. E, Quantification of colonies in the clonogenic assay described in D. F, Representative images of some of the tumors obtained in a mouse MCF-7 tumor xenograft after treatment with vehicle and X15695. G, Tumor volumes of established subcutaneous MCF-7 xenografts treated daily via oral gavage with vehicle or X15695 (30 mg/kg body weight). The values are the mean ± SEM (*, P = 0.05; n = 14). H, Tumor weights at the end of treatment were recorded and presented in the bar chart. The values are the means ± SEM (*, P = 0.05; n = 14). I, Mouse body weights measured twice weekly for all animals over 16 days. Presented are the mouse body weight changes, in percentages, for each group. J, Western blots analysis of ERα and p53 from the extracts of select tumors at the end of treatment. Anti-vinculin antibody was used to determine the level of protein loaded. Quantification of ERα (K) and p53 (L) protein levels in select tumors relative to the vinculin loading control. The results are the mean ± SEM. *, P ≤ 0.05; ***, P ≤ 0.001; n = 12.

X15695 reactivates p53. A, Western blot analysis of mortalin and p53 in immunoprecipitated extracts of MCF-7 cells treated with 1 μmol/L X15695 for 48 hours and lysed. 50 μg of cell extract was used as input control while 1,000 μg was immunoprecipitated with anti-p53 or IgG antibodies immobilized on protein A/G agarose beads. B, Western blot analysis of mortalin and BAG1 in immunoprecipitated extracts of MCF-7 cells treated with 1 μmol/L X15695 for 48 hours. MCF-7 cells were treated and processed as in A with the exception that the immunoprecipitation was carried out with a mouse anti-BAG1 antibody. C, Western blot analysis of BAG2 and BAG5 in immunoprecipitated extracts of T47D cells treated with 1 μmol/L X15695 for 48 hours. A total of 70 μg of cell extracts was used as input control while 1,000 μg was immunoprecipitated with anti-p53 or IgG antibodies immobilized on protein A/G agarose beads. D, Western blot analysis of p53 protein after transfection of MCF-7 and T47D cells with control and p53 siRNA and treatment with vehicle or X15695 (1 μmol/L) for 24 hours. β-actin was used for the loading control. E, Cell viability determination using MTT assay in control and p53 siRNA transfected cells treated with vehicle or X15695 (1 μmol/L) for 72 hours (n ≥ 3; ***, P ≤ 0.001; *, P ≤ 0.05). Cell-cycle analysis of MCF-7 (F) and T47D (G) cells treated with vehicle or 1 μmol/L X15695 for 48 hours. A total of 1 × 106 cells were stained with 7-AAD and used for flow cytometry measurement. Data were analyzed by FlowJo software. Apoptosis assessment of MCF-7 (H) and T47D (I) cells treated with 1 μmol/L X15695 for 48 hours. The cells were double stained with 7-AAD and Annexin V. Cells stained with 7-AAD or Annexin V alone were used for compensation of double staining. A total of 1 × 106 cells were used for the flow cytometry measurement. The quantified results are the mean ± SEM (n = 3; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.0001; ns is non-significant).

Volcano plots and Hallmark gene set analyses showing pathways targeted by X15695.

X15695-mediated regulation of expression and cellular localization of p53 in prostate cancer cells.

Supplementary data and Supplementary figure legends.

Crystallographic data and structure refinement information for X15695, X15696 and X19168.

X15695 attenuates ERα-mediated target gene expression. GSEA plots of the top gene sets identified in a comparison of E2 + X15695 versus E2 (A) and X15695 versus vehicle (B) in MCF-7. C–H, Quantitative RT-PCR to detect the effect of X15695 on the expression of the indicated p53 target genes in MCF-7 cells in the presence and absence of E2. Cells were treated with the indicated concentrations of X15695 in the presence and absence of 10 nmol/L E2 for 16 hours. The data represent the mean ± SEM (n = 4; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001; ns, not significant).

X15695-mediated attenuation of ERalpha target gene expression and reactivation of p53.